74 kilodalton outer membrane protein from moraxella catarrhalis

ABSTRACT

A protein from the  M. catarrhalis  designated the 74 kD protein is isolated and purified. The 74 kD protein has an amino-terminal amino acid sequence which is conserved among various strains of  M. catarrhalis . The protein has a molecular weight of approximately 74,9 kD as measured on a 10% SDS-PAGE gel, while its molecular weight as measured by mass spectrometry is approximately 74 kD. The 74 kD protein is used to prepare a vaccine composition which elicits a protective immune response in a mammalian host to protect the host again disease caused by  M. catarrhalis.

This application claims priority from PCT application, PCT/US98/01840,filed Jan. 29, 1998, which claims priority from provisional applicationSer. No. 60/036,827, filed Jan. 31, 1997, the entire disclosures ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to an approximately 74,000 Dalton (74 kD) outermembrane protein purified from Moraxella catarrhalis.

BACKGROUND OF THE INVENTION

Moraxella (Branhamella) catarrhalis is one of the major bacterialpathogens causing otitis media in children (Bibliography entries1,2,3,4). It also causes sinusitis, laryngitis, tracheitis, pneumonia,and other respiratory diseases in children and adults (5,6,7). Aprophylactic vaccine is clearly needed because nearly all clinicalisolates are resistant to β-lactam antibiotics (8,9).

The outer membrane proteins (OMPs) of M. catarrhalis are beinginvestigated as potential vaccine candidates because they are readilyaccessible to antibodies. Indeed, antibodies elicited in mice towardscertain OMP's including UspA and the CopB have already been shown tohave biological activity, such as bactericidal activity, adhesionblocking activity and enhanced pulmonary clearance of the bacteria in ananimal model (10,11,12,13). Serology data from humans who have suffereda recent M. catarrhalis infection indicates that humans developantibodies towards OMP's following natural infection (14,15,16,17,18).This suggests that OMP's are the targets of the host's defensemechanisms. UspA-specific antibodies are present in normal human serumand these antibodies have bactericidal activity. There are also highlevels of antibodies towards OMPs of approximately 80 kD in sera fromboth healthy humans and patients recovering from recent M. catarrhalisinfections (16). Several proteins from M. catarrhalis migrate withinthis size range. Among them are CopB (12), the B1 protein (18), atransferrin binding protein (TbpB) and a lactoferrin binding protein(LbpB) (19) Whether these proteins are the same or different from oneanother has yet to be determined. None of them, however, has beenevaluated in a purified form for vaccine use.

An efficacious vaccine to protect against diseases caused by M.catarrhalis should confer protection at all stages of disease. Thesestages include bacterial colonization on mucosal surfaces, bacterialmultiplication, spread and invasion, and the development of inflammatoryresponse. Multiple bacterial components may be required to formulate anefficacious vaccine. Although there is pre-clinical data to suggest thatsome surface components of M. catarrhalis are potential vaccineantigens, it is as yet unclear if these components will confersufficient protective immunity in humans. Thus, it is important toidentify and evaluate new bacterial antigens for vaccine use.

SUMMARY OF THE INVENTION

Accordingly, it is an object of this invention to isolate, purify andcharacterize an additional protein from M. catarrhalis. It is a furtherobject of this invention to test whether this protein is a viablevaccine candidate in appropriate model systems.

These and other objects of the invention as discussed below are achievedby the isolation and purification of a protein from M. catarrhalis whichis designated the 74 kD protein, based on approximate molecular weightas measured by mass spectrometry, as well as peptides of the 74 kDprotein comprising an epitope or epitopes thereof. The isolated andpurified 74 kD protein from M. catarrhalis has an amino-terminal aminoacid sequence which is conserved among various strains of M.catarrhalis. This amino-terminal amino acid sequence comprises thesequence Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr (SEQ IDNO:1), where the first residue is not identified, or a biologicallyequivalent amino-terminal amino acid sequence thereof. The protein ofthis invention has a molecular weight of approximately 74.9 kD asmeasured on a 10% SDS-PAGE gel, while its molecular weight as measuredby mass spectrometry is approximately 74 kD.

In another embodiment of this invention, the isolated and purified 74 kDprotein or a peptide of the 74 kD protein comprising an epitope orepitopes thereof, is used to prepare a vaccine composition which elicitsa protective immune response in a mammalian host. The vaccinecomposition may further comprise an adjuvant, diluent or carrier.Examples of such adjuvants include aluminum hydroxide, aluminumphosphate, MPL™, Stimulon™ QS-21, and IL-12. The vaccine composition isadministered to a mammalian host in an immunogenic amount sufficient toprotect the host against disease caused by M. catarrhalis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the characterization of the purified 74 kD protein fromO35E strain by SDS-PAGE and Western blots.

FIG. 1A depicts a Coomassie blue stained 4-15% SDS-PAGE showing enriched74 kD protein eluted from the S column (lane 2) and hydroxyapatitecolumn (lane 3). The molecular weight standards (lane 1:200 (myosin);160 (β-galactosidase); 97 (phosphorylase); 66 (serum albumin); 45(ovalbumin); 31 (carbonic anhydrase); 21 (trypsin inhibitor); 14(lysozyme)) are in thousands.

FIG. 1B depicts purified 74 kD protein by silver staining; the loadingwas 3 μg (lane 1), 15 μg (lane 2) and 30 μg (lane 3).

FIG. 1C depicts western blots detected with either mouse antiserumagainst the purified 74 kD protein (lane 1) or MAb 72-32 (lane 2).

FIG. 2 depicts the expression level of 74 kD protein by O35E strainunder different culture conditions. Bacteria were cultured in regularbrain heart infusion (BHI), BHI supplemented with 10 mMethylenediaminediacetate (EDDA) or 100 μM iron. Bacterial lysatesapplied to the membrane were detected by MAb 72-32 in dot blot andwestern blot assays.

FIG. 3 depicts the expression of transferrin binding proteins by O35Estrain under different culture conditions. Samples on the membrane werebacterial lysates as described for FIG. 2. Transferrin binding by thepurified 74 kD protein is shown in the fourth column.

FIG. 4 depicts the variation in the expression level of 74 kD protein byM. catarrhalis strains. Bacterial lysates from seven strains of M.catarrhalis were titrated in 3-fold dilutions in a dot blot and detectedwith mouse polyclonal antibodies against the purified 74 kD protein fromO35E strain (1:200).

FIG. 5 depicts the reactivity of the mouse anti-74 kD serum toheterologous strains of M. catarrhalis. Bacterial lysates containing 10μg of total protein were resolved in 4-15% SDS-PAGE and tested in awestern blot with mouse antiserum against the purified 74 kD proteinfrom O35E strain (1:5,000). Lanes 2-10 are strains O35E, TTA24,ATCC25238, 324-171, 128-179, 430-345, 111-210, 219-96, and 1230-359,respectively. The molecular weight standards (lane 1) are 133 kD(β-galactosidase) and 71 kD (bovine serum albumin).

FIG. 6 depicts the Western blot analysis of mouse antisera raisedagainst the 74 kD protein from TTA24 strain (bottom) or pooled 74 kDproteins from strains TTA24 and 430-345 (top). Each lane was loaded with10 μg of whole bacterial lysates or 1.5 μg of purified 74 kD protein.Antisera were from week 6 bleeding diluted 1:1,000.

FIG. 7 depicts the presence of antibodies to the 74 kD protein in normalhuman sera. Purified 74 kD protein from O35E strain was reacted withfive serum samples (Lanes 2-6) from healthy adults in a western blot.The molecular weight standards shown in lane 1 are the same as thosedescribed in FIG. 5.

FIG. 8 depicts the reactivity of anti-74 kD protein antibodies purifiedfrom adult serum to the whole bacterial lysates. O35E strain lysatescontaining 10 μg of protein were reacted with affinity purified humanantibodies against the 74 kD protein in a western blot (lane 2). Themolecular weight standards shown in lane 1 are the same as thosedescribed in FIG. 5.

FIG. 9 depicts the inhibition of transferrin binding by antibodies tothe 74 kD protein. Various amounts of O35E lysate were spotted on anitrocellulose membrane. The membrane was incubated with PBS (panel A);1:100 diluted normal mouse serum (panel B); anti-serum to the 74 kDaprotein from O35E strain (panel C); or control anti-serum to the UspA(panel D). The membranes were then probed with biotin-labeledtransferrin.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to an isolated and purified M. catarrhalisprotein designated the 74 kD protein. This 74 kD protein has anamino-terminal amino acid sequence which is conserved among the threestrains examined of M. catarrhalis. The protein of this invention has amolecular weight of approximately 74.9 kD as measured on a 10% SDS-PAGEgel, while its molecular weight as measured by mass spectrometry isapproximately 74 kD. The amino acid composition of the protein has alsobeen determined. The invention relates further to peptides of the 74 kDprotein comprising an epitope or epitopes thereof. Such peptidesincorporate one or more epitopes that are immunologically cross-reactivewith one or more epitopes of the 74 kD protein. Such peptides are firstgenerated and then tested for cross-reactivity.

Initially, the 74 kD protein was purified from salt wash vesicles madefrom O35E strain and evaluated in mice. Preliminary results indicatedthat the 74 kD protein is surface exposed, in that the mouse antiserahad bactericidal activity, and immunized mice exhibited enhancedclearance of the M. catarrhalis in a murine challenge model. However,this particular 74 kD preparation was contaminated withlipooligosaccharide (LOS) which also induced an antibody response inmice. Subsequently, the 74 kD protein was purified from three strains ofM. catarrhalis by a modified procedure. These preparations lackeddetectable LOS or other protein contamination. Example 1 below describesthe new purification method, which involves the extraction of theprotein directly from whole bacterial cells from M. catarrhalis strainsO35E and 430-345, followed by a series of column chromatography steps. Amodified version of this method was also used to purify the 74 kDprotein from TTA24 strain.

The 74 kD protein migrates slightly more slowly on a 4-15% gradientSDS-PAGE than CopB, another M. catarrhalis protein (data not shown).SDS-PAGE analysis using a 10% w/v acrylamide gel provided an estimatedmolecular weight for this protein of approximately 74.9 kD (see Example4). The actual molecular mass as determined by mass spectrometry wasapproximately 74 kD (see Example 4). The N-terminal sequences from O35E,TTA24 and 430-345 strains were found to be identical to the extent ofsequencing of these strains, that is, for the first 18 residues (seeExamples 6 and 7).

Two alternative methods for the purification of the 74 kD protein werealso employed. For both methods, the salt wash vesicles, prepared aspreviously described (11), were suspended in a 0.5% Triton X-100™ and 10mM N-[hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid] (HEPES) bufferand incubated for one hour at room temperature. The suspension wascentrifuged at low speed (10,000×g for 20 minutes) at 4° C. to removeparticulates. For the first method, the supernatant was passed over aDEAE Sepharose® (Pharmacia, Piscataway, N.J.) column equilibrated withthe same buffer. A single band of 74 kD protein, as seen by SDS-PAGE,eluted in the flow through and in the buffer wash, while contaminantseluted upon increasing the salt (NaCl) concentration. For the secondmethod, the supernatant was passed over a CM Sepharose® columnequilibrated with the same Triton X-100™-HEPES buffer. The proteins onthis column were then eluted with a step gradient of increasing saltconcentration. The 74 kD protein, which eluted at 200 mM of NaCl,migrated as a single band. Only two major outer membrane proteins fromsalt wash vesicles, the 74 kD protein and the CopB protein, migrate atabout this size on SDS-PAGE. The 74 kD protein failed to react bywestern blotting with a monoclonal antibody 10F3 specific for the CopBprotein. Minor amounts of the C/D protein present can be removed byusing an additional ion exchange column.

This invention also comprises polypeptides whose amino-terminalsequences differ from those of the 74 kD protein, but are biologicallyequivalent to those described for that protein. Such polypeptides may besaid to be biologically equivalent to the 74 kD protein if theirsequences differ only by minor deletions from or conservativesubstitutions to the amino acid sequence, such that the tertiaryconfigurations of the sequences are essentially unchanged from those ofthe 74 kD protein and biological activity is retained.

For example, alanine, a hydrophobic amino acid, may be substituted byanother less hydrophobic residue, such as glycine, or a more hydrophobicresidue, such as valine, leucine, or isoleucine. Similarly, substitutionof one negatively charged residue for another, such as aspartic acid forglutamic acid, or one positively charged residue for another, such aslysine for arginine, as well as changes based on similarities ofresidues in their hydropathic index, can also be expected to produce abiologically equivalent product. Each of the proposed modifications iswell within the routine skill in the art, as is determination ofretention of biological activity of the encoded products. Therefore,where the term “74 kD protein” is used in either the specification orthe claims, that term will be understood to encompass all suchmodifications and variations which result in the production of abiologically equivalent protein.

The N-terminus from the O35E strain and two internal peptides from achymotrypsin digest of the same protein were determined and found tohave no homology with any other known protein from M. catarrhalis insearches of the GenBank CDS translations, PDB, SwissProt, Spupdate andPIR databases with Basic Local Alignment Search Tool (BLAST) (20).However, the two internal peptides have significant sequence homology tothe transferrin binding protein from N. meningitidis and H. influenzae(see Example 8). The amino acid composition of the 74 kD protein is setforth in Example 5.

Purified 74 kD proteins from strains O35E and 430-345 were immunogenicin mice and their antibodies reacted with the homologous strain bywhole-cell ELISA; however, the whole-cell titers toward heterologousstrains varied considerably (see Example 12). The 74 kD protein fromTTA24 strain appeared to be better conserved, because antibodies made tothis protein exhibited moderately high reactivity to heterologousstrains by whole cell ELISA (Example 12). Antisera against the purifiedproteins from all three strains had complement-dependent bactericidalactivity toward all heterologous strains (see Example 9). This suggeststhat antibodies towards the conserved epitopes are important.

The level of antibodies reactive to heterologous strains andbactericidal antibody titers were improved by using certain adjuvantssuch as Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc., Worcester,Mass.) or a mixture of MPL™ (3-O-deacylated monophosphoryl lipid A; RIBIImmunoChem Research, Inc., Hamilton, Mont.) and aluminum phosphate (seeExamples 10 and 11). Mice immunized with the 74 kD proteins purifiedfrom strains O35E and 430-345 exhibited strong pulmonary clearance ofthe O35E strain toward which the antibodies reacted with high titers(P<0.01), but not the TTA24 strain toward which the sera reacted poorly(see Example 13). Further, normal human sera contain naturally acquiredantibodies towards the conserved epitopes on the 74 kD proteins fromboth O35E and TTA24 strains (Example 14). This suggests that M.catarrhalis expresses this protein in vivo and that the 74 kD protein isa target of the immune response upon natural infection. These humanantibodies reacted better to purified 74 kD protein from TTA24 strainthan protein from O35E strain (see Example 15). This suggests that the74 kD protein from TTA24 strain is conserved.

The relationship of the 74 kD protein to other proteins will now bedescribed. Eight major outer membrane proteins from M. catarrhalis wereinitially described by Bartos and Murphy, who designated these OMPs A-Hin the order of decreasing molecular weight (21). Two additional outermembrane proteins, designated UspA (13) and B1 (18), were describedlater. The OMP B was renamed B2 after the discovery of B1 (18). Exceptfor their similarity in molecular mass, the B1 and B2 proteins areunrelated. OMP B2 is probably the same protein as CopB described byHelminen et al. (12). This protein has a molecular mass of 82 kD, andappears to play a role in iron acquisition (22).

The 74 kD protein described in this application is distinct from theCopB protein in two aspects. First, a monoclonal antibody (MAb)designated 72-32, which reacts with the 74 kD protein, did not reactwith a recombinant CopB made in E. coli on western blot (data notshown). Nor did a CopB specific MAb, 10F3, react with the purified 74 kDprotein in the same assay. Second, the N-terminal amino acid sequenceand two internal peptide sequences of the 74 kD protein were not foundin the predicted protein sequence deduced from the published genesequence of CopB (12).

The 74 kD protein of this invention is most similar to the B1 protein interms of size and the degree of antigenic conservation. The OMP B1 wasinitially described by Sethi et al. (18), who did not isolate or purifythe protein. Sethi et al. noted the consistent presence of polyclonalantibodies towards an 74 kD minor protein in sera from patients withbronchiectasis. They also reported that B1 protein had surface exposedepitopes and appeared to be antigenically variable among strains of M.catarrhalis. The level of B1 expression was up-regulated underiron-limiting culture conditions for some isolates (23). There are alsoreports that the B1 protein binds transferrin (24). These reportssuggested that the B1 protein may be involved in iron acquisition andutilization. The usefulness of the B1 protein as a vaccine antigen wasnot investigated in these reports. In contrast, the data presented inthis application did not show significant changes in 74 kD proteinexpression levels by either depleting or supplementing iron in theculture broth (see Example 3).

Like B1 protein, the 74 kD protein binds transferrin (see Example 3).Transferrin-binding proteins have been detected in several bacterialspecies, including M. catarrhalis. Using transferrin and lactoferrinaffinity columns, Bonnah et al. identified two distinct receptorproteins from M. catarrhalis with molecular weight masses ofapproximately 80 kD (19). This is the same range as for the proteinsdesignated B1 and CopB. Unfortunately, these reports did not provideenough information on the biochemical, immunological and molecularaspects of these proteins to allow the identification of the 74 kDprotein as either of these proteins. However, many of the properties ofthe TbpB from the Neisseria family and Haemophilus influenzae aresimilar to those of the 74 kD protein from M. catarrhalis. These includethe ability to bind transferrin in both native or denatured form andantigenic heterogeneity.

However, the 74 kD protein of M. catarrhalis differs from the TbpBprotein of Neisseria in several respects. First, the molecular weight ofthe 74 kD protein is relatively well conserved from strain to strain,while the molecular weight of TbpB in Neisseria varies from strain tostrain (25). Second, mouse antibodies made against the 74 kD proteinfrom TTA24 strain reacted with all strains of M. catarrhalis by wholecell ELISA, and were bactericidal toward all strains assayed. Incontrast, antibodies to TbpB of Neisseria reacted only with a fractionof strains by ELISA and were only bactericidal toward strains to whichthe antibodies bound (26). Finally, the expression level of the 74 kDprotein appears to be constitutive, while TbpB (from both Neisseria andHaemophilus), like the B1 protein, is iron repressed. Therefore, withoutfurther information, it is unclear whether the 74 kD protein, the B1protein and TbpB protein are all the same protein.

Having isolated and characterized the 74 kD protein, the next step is toevaluate its potential as a vaccine antigen. Several lines of evidencepresented in this application indicate that the 74 kD protein is apotential vaccine antigen candidate. First, as detailed in Example 2below, it contains surface exposed epitopes. This is important, becauseonly surface exposed epitopes are accessible to the antibodies. Theepitope recognized by the MAb 72-32, although not conserved among allisolates, is surface exposed. Further, both the antibodies toward the 74kD protein purified from the human sera and antibodies made to thepurified protein in mice reacted with several strains of M. catarrhalisby whole cell ELISA. Some of the epitopes are clearly conformational,since many of the monoclonal antibodies react with purified 74 kDprotein and whole bacterial cells by ELISA, but do not react with thedenatured 74 kD protein on western blot. It also suggests that thepurified protein retains at least some conformational epitopes.

Second, some surface exposed epitopes of the 74 kD protein are conservedamong strains of M. catarrhalis. An efficacious vaccine needs to conferprotective immunity against most, if not all, strains. Because theexpression of the protein appears to vary from isolate to isolate invitro (see Example 3), the degree of antigenic variation of the 74 kDprotein among M. catarrhalis strains has been difficult to assess.Nevertheless, it is evident that the 74 kD protein contains conservedepitopes. Antibodies to the 74 kD protein, whether produced in mice ordeveloped in humans following natural infection, were bound by wholebacterial cells of the heterologous strains. The 74 kD protein fromTTA24 strain is fairly conserved, whereas the 74 kD protein from twoother strains studied is either less conserved or the strain-specificepitopes are more immunogenic than the conserved epitopes. Current dataseems to suggest that the 74 kD protein from TTA24 strain may have morepotential as a vaccine antigen because it elicits highly cross-reactiveantibodies toward heterologous strains (see Example 12, Table 9).

Third, antibodies toward conserved surface epitopes elicited by thepurified protein were bactericidal (see Example 9). Although it isunclear how antibodies mediate protection against infections by M.catarrhalis, they could play a role in a number of pathways. Theseinclude inhibition of bacterial adherence, interference of nutrientuptake, opsonic phagocytosis and complement-dependent killing. It wasobserved that adults, a population usually resistant to M. catarrhalisinfections, have a significantly higher level of serum bactericidalactivity to M. catarrhalis than children, a population susceptible to M.catarrhalis infections (data not shown). The finding that mouse antiserato the 74 kD protein exhibit bactericidal activity toward heterologousstrains in a antibody concentration-dependent manner suggests that thisprotein is a protective antigen. Clearly, the conserved epitope of the74 kD protein is an important target of the bactericidal antibodies.

Finally, mice immunized with the 74 kD protein exhibited enhancedpulmonary clearance of the bacteria in the murine challenge model (seeExample 13). Although the mechanisms of bacterial clearance in thismodel are unknown, antibodies clearly play an important role in thismodel (27). In addition to the 74 kD protein described in thisapplication, UspA and CopB have been shown to promote enhanced clearancein this model (11,12,13). Further, only antibodies to certain epitopesof these proteins appear to enhance bacterial clearance. Thus, the modelhas been useful for selecting vaccine candidates that may elicitantibodies with in vivo biological activity. Two strains of the bacteriaare suitable for challenge in the murine pulmonary model, and both weretested. Enhanced bacterial clearance was seen for O35E strain whichexhibited strong reactivity with the antibodies against the 74 kDprotein prepared from two different strains. Homologous clearance ofstrain TTA24 was seen; however, heterologous clearance was not observed.This is probably because of the poor antibody reactivity toward thisisolate elicited by the purified protein. It remains to be determinedwhether the 74 kD protein from TTA24 strain will promote enhancedclearance of heterologous strains in this challenge model.

Another potential mechanism by which the 74 kD protein can conferprotection in humans is by eliciting antibodies that block iron uptakeby M. catarrhalis. Iron is an essential element for the bacteria to growand cause pathogenesis in vivo. However, the concentration of free ironin the extracellular environment is too low to support bacterial growth.Extracellular iron is virtually all sequestered in host proteins such aslactoferrin on the mucosal surface and transferrin in the serum. Severalbacterial species including M. catarrhalis can acquire iron from hostproteins through their specialized surface receptors, such astransferrin binding proteins (TbpA and B) and lactoferrin bindingproteins (Lbp A and B). To obtain iron, TbpB, a lipoprotein that is anouter membrane protein, functions as a receptor for transferrin in bodyfluids.

It has been shown that M. catarrhalis can utilize transferrin as thesole iron source for growth in vitro (19), and this may be an importantmechanism of iron acquisition in vivo. The mechanism by which M.catarrhalis acquires iron from transferrin is not clear; however, itvery likely requires direct interaction of Tbp with transferrin. Example16 indicates that antibodies to the 74 kD protein from M. catarrhalisare able to specifically block transferrin binding by the bacteriallysates. This is consistent with previous findings that antibodies tothe meningococcal TpbB were able to lower the growth rate ofmeningococci when human transferrin was the sole iron source (28).

The transferrin binding proteins from other species are reported to belipoproteins (29,30). This usually blocks the protein's N-terminus,preventing the determination of the N-terminal sequence (31). Because anN-terminal sequence could be determined for the 74 kD protein from threestrains, this represents another possible difference between thetransferrin binding protein and the 74 kD protein of M. catarrhalis.

Therefore, the 74 kD protein and peptides of the 74 kD proteincomprising an epitope or epitopes thereof are useful in the preparationof vaccines to confer protection to humans against otitis media andother diseases caused by M. catarrhalis.

These vaccine compositions comprise the isolated and purified 74 kDprotein of M. catarrhalis or a peptide of the 74 kD protein comprisingan epitope or epitopes thereof, wherein the vaccine composition elicitsa protective immune response in a mammalian host.

Vaccines containing the 74 kD protein or peptides may be mixed withimmunologically acceptable diluents or carriers in a conventional mannerto prepare injectable liquid solutions or suspensions. In addition, thevaccines may include aluminum hydroxide, aluminum phosphate (alum) orother pharmaceutically acceptable adjuvants, such as Stimulon™ QS-21(Aquila Biopharmaceuticals, Inc. Worcester, Mass.), MPL™, and IL-12(Genetics Institute, Cambridge, Mass.).

The vaccines of this invention may also include additional M.catarrhalis proteins which are known in the art. Examples of suchproteins are those designated CopB, UspA, C/D and E.

The vaccines of this invention further include other protective agentswhich are coupled to the 74 kD protein or peptides, such that the 74 kDprotein or peptides function as a carrier molecule. For example, agentswhich protect against other pathogenic organisms, such as bacteria,viruses or parasites, are coupled to the 74 kD protein or peptides toproduce a multivalent vaccine useful in the prevention of both M.catarrhalis infection and other pathogenic infections. In particular,the 74 kD protein or peptides can serve as immunogenic carriers by beingconjugated to Haemophilus, meningococcal or pneumococcal polysaccharidesor oligosaccharides. In addition, the 74 kD protein or peptides arecoupled to another antigenic moiety of M. catarrhalis such aslipooligosaccharides.

The vaccines of this invention are administered by injection in aconventional manner, such as subcutaneous, intraperitoneal orintramuscular injection into humans, as well as by oral administrationand intranasal administration, to induce an active immune response forprotection against otitis media caused by M. catarrhalis. The dosage tobe administered is determined by means known to those skilled in theart. Protection may be conferred by a single dose of vaccine, or mayrequire the administration of several booster doses.

Normally, in the absence of human clinical data, active immunization ina recognized animal model is relied upon to predict the efficacy of avaccine in humans. Here, the pulmonary clearance is measured in themurine challenge model. The murine challenge model permits an evaluationof the interaction of M. catarrhalis with the lower respiratory tract,as well as an assessment of pathologic changes in the lungs (32,33).This model reproducibly delivers an inoculum of bacteria to a localizedperipheral segment of the murine lung. Bacteria multiply within thelung, but are eventually cleared as a result of host defense mechanismsand the development of a specific immune response.

In the present invention, the 74 kD protein is shown to be a viablevaccine candidate both because antibodies elicited by the 74 kD proteinwere bactericidal and because mice immunized with the 74 kD proteinexhibited enhanced pulmonary clearance of M. catarrhalis in the murinechallenge model.

The 74 kD protein or peptides thereof are also useful to producepolyclonal antibodies for use in passive immunization against M.catarrhalis. Polyclonal antisera are generated from animals immunizedwith the 74 kD protein or peptides thereof.

The 74 kD protein or peptides thereof are further used to generatemonoclonal antibodies which may be used to diagnose the presence of M.catarrhalis in a clinical sample or a laboratory strain. The monoclonalantibodies react with M. catarrhalis, but not with other bacteria.

In order that this invention may be better understood, the followingexamples are set forth. The examples are for the purpose of illustrationonly and are not to be construed as limiting the scope of the invention.

EXAMPLES Example 1 Purification and Characterization of the 74 kdProtein

Purification

The 74 kD protein was purified from strains O35E and 430-345 using thesame procedure. The bacteria were grown in Fernbach flasks containing1.3 liters of CY broth (10 g casamino acids, 15 g yeast extract perliter of distilled water) at 37° C. for 22 hours with shaking at 200rpm. The bacteria were harvested by centrifugation (10,000×g for 20minutes), resuspended in 50 ml of phosphate buffer (10 mM, pH 6.0)containing 0.1% Triton X-100™ (J.T. Baker Inc., Philipsburg, N.J.), andstirred for one hour at room temperature (RT). Particulates were removedby centrifugation (10,000×g, 60 minutes) and the soluble extract loadedon a 5 ml bed volume column of S Sepharose (Pharmacia, Piscataway,N.J.). The column was eluted with a NaCl step gradient in 10 mMphosphate buffer (pH 6.0) containing 0.1% Triton X-100™. The 74 kDprotein was detected by dot blotting using MAb 72-32. Enriched fractionsof the 74 kD protein (which eluted between 70-210 mM NaCl) were pooled,and applied to a 3 ml bed volume column of hydroxyapatite (BIO-RADLaboratories). The column was washed with 10 mM phosphate buffer (pH6.0) and eluted using a step gradient of phosphate buffer (pH 6.0). The74 kD enriched fractions were pooled, concentrated using aCentriprep®-30 (Amicon, Beverly, Mass.), and passed over a column(2.6×100 cm) of Ultrogel AcA 44 (BioSepra Inc., Marlborough, Mass.) at aflow rate of 1.0 ml per minute in PBS (pH 7.4). The proteinconcentration was determined by a micro-bicinchoninic acid assay(Micro-BCA) (Pierce, Rockford, Ill.).

The 74 kD protein was purified from strain TTA24 by a slightly differentprocedure. The initial attempt to purify the 74 kD protein from TTA24strain cultured in CY broth by the above method did not yield anyprotein. It was then observed that TTA24 cultured on Mueller-Hinton agarplates expressed a higher level of 74 kD protein. So, the bacteria grownon these plates were used as the starting material for purification. Theprotein did not bind to the S Sepharose® column under conditions usedfor the 74 kD protein from O35E. Instead, it was purified by sequentialpassage over first a hydroxyapatite column, then a Q High Performancecolumn (Pharmacia) and finally an Ultrogel AcA44 column.

Characterization

The 74 kD protein was associated with bacterial cells cultured in CYbroth. It was readily extracted in phosphate buffer (10 mM, pH 6.0)containing 0.1% Triton X-100™ after 1 h stirring at RT. The 74 kDproteins from strains O35E and 430-345 were among the few proteins inthe whole cell extract that bound to the S column, and accounted for50-70% of the total protein in the fractions that were eluted with 10 mMphosphate buffer (pH 6.0) containing 70-210 mM NaCl. It exhibited strongbinding to a hydroxyapatite column and eluted with 500 mM phosphatebuffer containing no detergent. The bulk of the contaminants were in theflow through fraction off this column. The major peak eluted off theAcA44 size-exclusion column contained a single homogenous band with amolecular mass of 74 kD on a Coomassie blue stained 4-15% acrylamidegradient SDS-PAGE.

The 74 kD protein from TTA24 strain was enriched by the HA column. Itwas among the few proteins which did not bind the Q column and wasfinally purified by the size exclusion column. The yield of purifiedproteins, as shown in Table 1 below was approximately 1-3 mg from 1.3liter of broth culture.

TABLE 1 Purification of the 74 kD protein Bacterial Final yield strainCulture volume (mg) O35E 15 L 24   O35E 20 L 52   430-345  4 L  4.48TTA24 20 plates (15 6.3 cm diameter) (2 L)

The purified 74 kD proteins were analyzed by 4-15% gradient SDS-PAGEstained with Coomassie blue and silver staining. Reactivity tomonoclonal antibodies and polyclonal mouse serum was determined bywestern blot (FIG. 1).

Example 2 Monoclonal Antibodies

The MAbs toward the 74 kD protein were made using the procedure of Chenet al. (11). In summary, mice (BALB/c) used for the fusion wereimmunized with outer membrane vesicles made from M. catarrhalis O35Estrain. Hybridomas were first screened by ELISA against O35E wholebacterial cells. Those recognizing O35E whole cells were then tested forreactivity with purified 74 kD protein of the O35E strain by both ELISAand Western blotting. Reactivity toward heterologous strains wasdetermined by whole-cell ELISA. The selected hybridomas were cloned bylimiting dilution.

Two of the MAbs recognized the purified 74 kD protein by both ELISA andwestern blot. They did not react with the CopB protein. When the MAbsdesignated 72-32 and 81-8 were tested against six other M. catarrhalisstrains by western blot analysis, they only reacted with the homologousisolate O35E. Thus, these two MAbs recognize strain specific epitopes.The lack of reactivity to heterologous strains was confirmed by wholecell ELISA.

Thereafter, all parent clones from that fusion were screened by ELISA. Adozen clones had reactivity to both the purified 74 kD protein and O35Ewhole bacterium cells, but none reacted to 11 heterologous strains. Onlyfive of the 12 parent clones exhibited reactivity to the 74 kD proteinon western blot. The non-reactors are probably directed against theconformational epitopes.

These data indicated that the 74 kD protein has surface exposedepitopes, and some of them may be conformational. It also indicated thatthe 74 kD protein from O35E strain is either antigenically heterogeneousor the strain-specific epitopes are more immunogenic than the conservedepitopes.

Example 3 Expression Level and Transferrin Binding Assay

There are indications that B1 protein (18) binds transferrin and it maybe transferrin binding protein B (TbpB) (19). The 74 kD proteindescribed herein appears to have similar molecular mass as well as theantigenic heterogeneity typical of TbpB.

An initial test was performed to determine whether the purified 74 kDprotein could bind transferrin. This was determined by probing purified74 kD protein from strain O35E which was spot blotted on anitrocellulose membrane with biotin-labeled transferrin. Strongreactivity was detected (see FIG. 3). Thus, transferrin binding is acommon property of the 74 kD protein, B1 and TbpB. Since the expressionlevel of transferrin binding proteins B1 and TbpB of M. catarrhalis isreported to depend on the iron content of the culture medium (18,19),the next step was to determine if the 74 kD expression level could beincreased by depleting the iron in the culture broth. The methods usedto deplete iron included both phosphate precipitation and chelation withEDDA. The 74 kD protein from the whole cell lysates was quantitated bydot blot and western blot using MAbs. There appeared to be noappreciable change in the expression level of the 74 kD protein iniron-depleted culture (see FIG. 2). However, the level of thetransferrin binding proteins as determined by probing withbiotin-labeled transferrin in a dot blot assay significantly increasedin iron depleted culture (see FIG. 3). It is unknown whether thisincrease reflects changes in the ThpA or TbpB expression level. However,these preliminary results on the 74 kD expression level under ironlimiting conditions are not consistent with the published reports for B1and TbpB.

While the iron level did not appreciably affect expression, growth indifferent media did affect it. Lysates of bacteria adjusted to the sameabsorbance at 550 nm made from several strains of M. catarrhaliscultured in broth or on agar plates were probed with polyclonalantibodies against the purified 74 kD protein in a western blot. Thepolyclonal antibodies had been generated by immunizing mice with the 74kD protein which had been purified from O35E strain. The 74 kDexpression level appeared to be higher when the bacteria were culturedon an Mueller-Hinton agar plate than when cultured in broth culture(data not shown).

The expression level of 74 kD protein appears to be strain dependent.When a low dilution of antiserum against 74 kD protein from O35E strain(1:200) was used in a dot blot assay to react with serially dilutedwhole cell lysates from seven M. catarrhalis strains, the strongestreactivity was seen towards strains O35E, 120-345 and 430-345. Thereactivity toward the four other strains was three to nine fold less(see FIG. 4). Several attempts were made to purify the 74 kD proteinfrom the strains exhibiting low reactivity, and only small amounts ofthe protein appeared to be present in these strains.

Example 4 Molecular Weight of the 74 kD Protein

Determination of Molecular Weight by SDS-PAGE Analysis

The 74 kD protein purified from M. catarrhalis O35E was subjected toSDS-PAGE (10%, w/v, acrylamide) analysis (34) along with a wide-range ofprotein standards (Mark 12: apparent molecular weights of 200, 116.3,97.4, 66.3, 55.4, 36.5, 31, 21.5, 14.4, 6, 3.5 and 2.5 kD) obtained fromNovel Experimental Technology, San Diego, Calif. The gel was stainedwith Coomassie Brilliant Blue R-250. The destained gel was scanned usinga Personal Densitometer SI (Molecular Dynamics Inc., Sunnyvale, Calif.).The molecular weight of the purified protein, estimated using theFragmeNT Analysis software (version 1.1, Molecular Dynamics), was foundto be approximately 74.9 kD based on the molecular weight standards.

Determination of Molecular Weight by MALDI-TOF Mass Spectral Analysis

Accurate measurement of the molecular weight of the 74 kD protein wascarried out by Matrix Assisted Laser Desorption/IonizationTime-of-Flight (MALDI-TOF) mass spectrometry using a Lasermat™ 2000linear mass analyzer (Finnigan Mat Limited). The Lasermat™ uses thetechnique of matrix-assisted laser desorption (35) to ionize the sampleand Time of Flight to analyze the ions produced. The sample was embeddedin a matrix of 3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid) toenhance ionization of the sample. One microliter of the samplecontaining 5-10 pmol purified 74 kD protein was mixed with 1 μl of thematrix (10 mg/ml) dissolved in 70% (v/v) aqueous acetonitrile containing0.1% (v/v) trifluoroacetic acid. One microliter of this sample andmatrix mixture was loaded on a sample slide, allowed to dry andirradiated by a short pulse of UV light from a laser. Protein samplesusually generate a relatively simple spectra in this method, sinceprotein-related ions produced are predominantly of charge states z=+1[M+H]⁺ and z=+2 [M+2H]²⁺. Bovine serum albumin (catalog no. A0281, SigmaChemical Co., St. Louis, Mo.) of molecular weight 66,430.0 was used forexternal calibration.

The molecular weight of the 74 kD protein in the sample used for theSDS-PAGE analysis above was determined to be 73,987.7, while that of the74 kD protein purified from M. catarrhalis TTA24 was 73,793.6. Inaddition to the expected [M+H]⁺, the [M+2H]²⁺ and the [M+3H] ³⁺molecular ions of the 74 kD protein were also observed. Hence, it isreasonable to conclude that the molecular weight of the 74 kD protein isin fact approximately 74 kD, within the limits of experimental error.

Example 5 Amino Acid Composition Analysis

A sample of the 74 kD protein for amino acid analysis was hydrolyzed inglass tubes using 100 μl of 6 N HCl containing 5% phenol and 1%2-mercaptoethanol under vacuum for 22 hours at 110° C. The samples weresubsequently dried under vacuum followed by resolubilization in sampledilution buffer Na-S (Beckman Instruments, Inc., Fullerton, Calif.,U.S.A.). The amino acid composition was determined on a Beckman model6300 Amino Acid Analyzer (36) using a three step Na-citrate gradientaccording to manufacturer's instructions. The results were expressed asmol of residues per mol of the 74 kD protein based on a molecular weightof the 74 kD protein of 74,000. Cysteine and tryptophan residues werenot determined. Threonine and serine residues were not corrected fordestruction caused by the method of analysis used. Nine microliters ofsample (purified from M. catarrhalis O35E—salt wash vesicles) were drieddown and subjected to acid hydrolysis and subsequent amino acidanalysis. Results reported in Table 2 represent the mean of duplicatedeterminations.

TABLE 2 Amino Acid Composition of the 74 kD Protein residues Amino acidper mol Asp + Asn 104  Thr 58 Ser 44 Glu + Gln 67 Pro 27 Gly 77 Ala 56Val 35 Met  6 Ile 21 Leu 40 Tyr 25 Phe 28 His  8 Lys 72 Arg 21

Example 6 Amino (N-) Terminal Amino Acid Sequence Analysis

Purified 74 kD protein preparations were subjected to SDS-PAGE (34) todetermine homogeneity. Samples which contained traces of impurities weresubsequently subjected to electrophoretic transfer onto a polyvinylidenedifluoride membrane (ProBlott membrane, Applied Biosystems, Foster City,Calif.) using 10 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS),10% methanol (pH 11.0) as the transfer buffer (37). The membrane wasstained with Coomassie Brilliant Blue R-250 and the main bandcorresponding to the 74 kD protein was cut out. Amino-terminal proteinsequence analysis was carried out using an Applied Biosystems Model 477AProtein/Peptide Sequencer equipped with an on-line Model 120A PTHAnalyzer (Applied Biosystems). After the cleavage of each successiveamino-terminus, the anilinothiazolinone derivative formed was convertedto the more stable phenylthiohydantion (PTH) derivative by treatmentwith 25% trifluoroacetic acid at 64° C. for 20 minutes. The PTHderivatives were separated and identified on the PTH analyzer byreversed-phase HPLC using a Brownlee PTH C-18 column (particle size 5μm, 2.1 mm i.d.×22 cm l.; Applied Biosystems) with a modified twosolvent gradient system developed by the manufacturer (35). Thefollowing summarizes the results of N-terminal sequence analysis ofdifferent preparations of the 74 kD protein:

For the 74 kD protein purified from salt wash vesicles of M. catarrhalisO35E strain, approximately 18.7 μl of sample containing 20 μg ofpurified protein was subjected to SDS-PAGE followed by electroblottingand 20 cycles of N-terminal protein sequence analysis. The first 13residues were determined except for an unidentified peak at residue 1(SEQ ID NO:1):

Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr

1 5 10

For the 74 kD protein purified from a whole cell extract of M.catarrhalis O35E strain, approximately 7.41 μl of sample containing 20μg of purified protein was subjected to SDS-PAGE followed byelectroblotting and 25 cycles of N-terminal protein sequence analysis.The first 17 residues were determined except for an unidentified peak atresidue 1 (SEQ ID NO:2):

Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro  1               5                  10 Thr Pro Ile Pro Asn          15

For the 74 kD protein purified from a whole cell extract of M.catarrhalis TTA24 strain, approximately 14.3 μl of sample containing74.4 μg of purified protein was directly loaded in the sequencer and 30cycles of N-terminal protein sequence analysis performed. The first 20residues were determined except for an unidentified peak at residue 1(SEQ ID NO:3):

Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro  1               5                  10 Thr Pro Ile Pro Asn Ala Ser Gly         15                  20

The unidentified peak at residue 1 for each of the above samples wasidentical and followed the PTH-Glu peak by approximately 0.2 minutes.

Example 7 Extended N-Terminal Amino Acid Sequence Analysis

During N-terminal sequence analysis of the 74 kD protein, the abundanceof proline (Pro) residues was recognized to cause early termination ofthe sequence read. Pro residues are partially released during thestandard acid cleavage step in sequencing. Release of the remaining Prowith subsequent residues causes difficulty in sequence identification.In order to generate longer N-terminal sequences of the 74 kD protein,extended acid cleavage at Pro residues was carried out. This producedbetter results, but caused an increased amino acid background whichseemed to be due to the simultaneous sequencing of acid inducednon-specific cleavage products of the 74 kD protein. Chemical reductionof amino acid background build up during sequence analysis was carriedout at some of the cycles where proline residues occurred. This wasaccomplished by introducing O-phthalaldehyde, a reagent whichspecifically reacts with amino groups of all N-terminal primary aminoacids, without affecting corresponding prolyl residues, thereby blockingthe residual protein/peptide chains (background) from subsequentsequencing (39,40). Twenty milligrams of O-phthalaldehyde were dissolvedin 50 μl of 2-mercaptoethanol in 10 ml of acetonitrile and placed in theXl bottle in the sequencer. Twenty-six microliters of the 74 kD protein(a lot purified from M. catarrhalis O35E—whole cell extract) containing80.6 μg of protein was loaded on the sequencer. During sequencing,extended trifluoroacetic acid cleavage was carried out for Pro residuesat 9, 10, 12, 14 and 16, whereas O-phthalaldehyde treatment was carriedout for Pro residues at 9 and 16. This led to effective backgroundreduction and resulted in extended N-terminal sequence determination(the first 27 residues) of the 74 kD protein (SEQ ID NO:4):

Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro  1               5                  10 Thr Pro Ile Pro Asn Ala Ser GlySer Gly Asn Thr          15                  20 Gly Asn Thr  25

This sequence was analyzed using the BLAST alignment program to searchfor homology with other proteins. No significant homology was seen withany bacterial proteins in the previously listed data bases.

A similar procedure was used to obtain an N-terminal sequence for the 74kD protein from M. catarrhalis 430-345 strain. One and one-half nmol ofthe 74 kD protein from M. catarrhalis 430-345 strain was subjected toN-terminal protein sequence analysis using an Applied Biosystems Model477A Protein/Peptide Sequencer equipped with an on-line Model 120A PTHAnalyzer (Applied Biosystems), as described above. During sequencing,extended trifluoroacetic acid cleavage was carried out for the Proresidue at 14, whereas O-phthalaldehyde treatment was carried out forPro residues at 10 and 16. The first eighteen N-terminal residues weredetermined, again with the first residue not determined (SEQ ID NO:5):

Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro  1               5              10 Thr Pro Ile Pro Asn Ala          15

Example 8 Amino Acid Sequence Analysis of Internal Peptides

A sample of the 74 kD protein (1.5 mg of a lot purified from M.catarrhalis O35E—whole cell extract) was digested overnight at 37° C. inPBS with chymotrypsin (Boehringer Mannheim Biochemicals, Indianapolis,Ind.) using a substrate:enzyme ratio of 1:60 (w/w). Digestion wascomplete as adjudged by SDS-PAGE analysis. The digest was purified byreversed phase HPLC using a Vydac Protein C4 column (particle size 5 μm,0.46 cm i.d.×25 cm l; The Separations Group, Hesperia, Calif.). The HPLCconditions were as follows: flow rate 1.0 ml/min; Solvent A=0.1% aqueoustrifluoroacetic acid; Solvent B=acetonitrile: water, 80:20 (v/v)containing 0.1% (v/v) trifluoroacetic acid; linear gradient of 0-100%Solvent B over 45 minutes. Eluted peaks were detected by theirabsorbance at 220 nm and fractions were collected. The fractions weredried down and each resuspended in 50 μl water. Suitable fractions werepooled and aliquots were subjected to Tricine-SDS-PAGE (41) using 10-18%(v/v, acrylamide) gradient gels. The gels were stained with CoomassieBrilliant Blue R-250 followed by transfer on to polyvinylidenedifluoride membrane as stated above. Four different bands were cut outand subjected to N-terminal amino acid sequence analysis. Two of thepeptides, ‘fragment 1’ (SEQ ID NO:6) and ‘fragment 3’ (SEQ ID NO:8),generated essentially identical 14-residue sequences; the onlydifference was that the N-terminal residue was Thr for the former, whileGly was the predominant corresponding residue for the latter, although alower amount of Thr could be detected. Both of these sequencesoverlapped over four residues with the N-terminal sequence of anotherpeptide ‘fragment 2’ (SEQ ID NO:7) to generate a continuous stretch of25 amino acid residues. Since chymotrypsin usually cleaves proteins onthe carboxy terminal side of Trp, Tyr, Phe, Leu and Met residues, it isevident that partial cleavage of the Tyr¹⁰-Asn¹¹ bond in either of thepeptides ‘fragment 1’ or ‘fragment 3’ gave rise to the peptide ‘fragment2’. On the other hand, partial cleavage of the Tyr⁴-Gly⁵ bond in‘fragment 2’ permitted sequence information beyond this point to beobtained. Also, it was evident that both of the peptides ‘fragment 1’and ‘fragment 3’ were sequenced in their entirety.

Thr Asp Glu Lys Asn Lys Pro Asp Gly Tyr Asn Gly Glu Tyr  1               5                  10 (Fragment 1; SEQ ID NO:6)                            Asn Gly Glu Tyr Gly His Ser Ser Glu Phe ThrVal Asn Phe Lys                              1               5                  10                  15                            (Fragment 2; SEQ ID NO:7) Gly Asp Glu LysAsn Lys Pro Asp Gly Tyr Asn Gly Glu Tyr (Thr)  1               5                  10 (Fragment 3; SEQ ID NO:8)|---------------------------------25----------------------------------------------------|

In addition to the above, a fourth chymotryptic peptide (‘fragment 4’(SEQ ID NO:9)) gave the following sequence over twenty of its N-terminalresidues. The Thr at residue 20 could not be assigned with certainty.

Lys Ser Ile Val Ile Arg Asp Ala Asp Val Thr Gly  1               5                  10 Gly Phe Tyr Tyr Pro Asn Ala(Thr)          15                   20 (Fragment 4; SEQ ID NO:9)

These sequences were analyzed using the BLAST alignment program forcomparison with other proteins in the previously listed data bases. The25 amino acid peptide generated by the overlap of the fragments 1through 3 had homology with a conserved portion of the TbpB protein fromNeisseria meningitidis, as well as with a similar sequence fromHaemophilus influenzae. Fragment 4 was found to have homology with arepeated sequence in the TbpB protein from Neisseria meningitidis.

Example 9 Bactericidal Activity of Antiserum to the 74 kD Protein

A bactericidal assay was performed as described previously (11).Briefly, 50 μl of bacterial suspension (approximately 1,200 colonyforming units (CFUs) in PBS containing 1 mM CaCl₂ and 0.2 mM MgCl₂) weremixed, and incubated with 25 μl of antiserum against the 74 kD protein(same as used in Table 1) for 30 minutes at 4° C. Antiserum was testedat 3-fold dilutions starting from 1:50. Twenty-five μl ofimmunoglobulin-depleted human serum was then added as the complementsource, and the mixture incubated an additional 30 minutes at 35° C. Thenumber of remaining viable bacteria was determined by plating 50 μl ofthe assay mixture on Mueller-Hinton agar plates. The control consistedof bacteria, test sera and complement serum which had beenheat-inactivated at 55° C. for 30 minutes. Whole cell ELISA andbacteridal titers were determined based on pooled serum samples. Thepercentage of bacteria killed was calculated by the following formula: %killing=100×(CFU from the control—CFU from the sample)/CFU from thecontrol). The bactericidal activity of the antisera was expressed asbactericidal titer, i.e., the highest serum dilution resulting inkilling of 50% or more of the bacteria.

The mouse antisera raised against the 74 kD proteins in several studieswere tested in a bactericidal assay against seven M. catarrhalisstrains. BALB/c mice (10 animals/group, female, 6-8 week old at thebeginning of the study) were immunized at weeks 0 and 4 with 1 μg ofantigens mixed with 25 μg of Stimulon™ QS-21. The results are presentedin Table 3:

TABLE 3 Bactericidal (BC) activity of the anti-74 kD sera BC titer ofweek 6 sera to 74 kD protein from strain Assay O35E 430-345 strain Study1 Study 2 Study 3 Study 2* Study 3 O35E  <50    <50    <50     450 <50   TTA24  400  50  50   150 150 430-345 ND** 4,050   4,050   4,05012,150   ATCC25238  400 150  50   450  <50   125-114 1000 150 150 1,350450 216-96 1000  50 150 1,350 150 1230-359  <50   450 ND 1,350 ND*Strain 430-345 was not tested in Study 1. **ND = not done.

Sera against the 74 kD protein purified from strains O35E or 430-345exhibited killing of almost all strains (Table 3). The O35E strainappears to be resistant to bactericidal effects elicited by the 74 kDprotein of that strain. The bactericidal titers varied from strain tostrain and appeared to correlate with whole-cell ELISA titers (data notshown). For almost every strain assayed, the bactericidal titers werehigher for the antiserum against the 74 kD protein from strain 430-345than that against the O35E strain. This is consistent with wholecell-ELISA titers. Pre-immune sera from the same animals were notbactericidal.

Thus, antibodies to the 74 kD protein consistently exhibitedbactericidal activities against heterologous strains of M. catarrhalisin spite of low antibody titers by whole cell ELISA. This suggests thatthe bactericidal antibodies are directed toward the conserved epitopesof the 74 kD protein.

Antibodies against the 74 kD protein from strain TTA24 were bactericidaltowards all six strains assayed and the titers were >500. These resultsare presented in Table 4:

TABLE 4 Bactericidal titers of the week 6 serum from mice immunized with74 kD protein from TTA24 strain or with a mixture of 74 kD protein fromTTA24 and 430-345 strains Antisera to 74 kD Protein from Strains AssayTTA24 + strain TTA24 430-345 O35E 1,163     948 TTA24 >6,400   >6,400125-114 1,011   1,452 430-345 1,303 >12,800  216-96   587     3261230-359   555   1,181

Antisera to the mixture of 74 kD proteins from strains TTA24 and 430-345exhibited equivalent bactericidal titers against the heterologousstrains.

Example 10 Effect of Adjuvants on Whole Cell ELISA Titers

Several adjuvants were compared to determine if the selection ofadjuvant might augment the antibody response to the conserved epitopesof the 74 kD protein from O35E strain. Sera generated against 74 kDprotein from O35E strain were assayed against seven strains of M.catarrhalis by whole cell ELISA. In this assay, BALB/c mice (10animals/group, female, 6-8 week old at the beginning of the study) wereimmunized at weeks 0 and 4 with 1 μg of 74 kD protein. The doses ofadjuvants were: 25 μg for Stimulon™ QS-21, 50 μg for MPL™, and 100 μgfor aluminum phosphate (alum). Immune sera were made with proteinspurified from O35E strain. Whole cell ELISA titers were determined onpooled week 6 sera. The results are shown in Table 5:

TABLE 5 Reactivity of the antibodies elicited by 74 kD protein usingdifferent adjuvants whole cell ELISA titer to M. catarrhalis strainadjuvant O35E 430:345 TTA24 ATCC 125-114 216-96 1230 saline   102,274 17,023 195 160 2,271   270 1,091 QS-21 1,910,012 789,405 1,058  1,083   18,368   5,313 6,061 MPL 1,193,747 213,435 349 769 1,255   1221,639 Alum   235,736  33,117 484 300 1,488  2,643 1,185 MPL + Alum2,443,332 138,246 5,380   6,292   14,889  11,205 4,283

Based on titers to the homologous strain, it appeared that the adjuvantsStimulon™ QS-21, MPL™ and the MPL™-alum mixture all potentiated theimmunogenicity of the 74 kD protein to a similar degree, while aluminumphosphate did not appear to act as an adjuvant. When whole cell ELISAtiters against heterologous strains were examined, only 74 kD proteinadjuvanted with Stimulon™ QS-21 or MPL™-alum elicited significant titersof antibodies. Mice immunized with 74 kD protein and MPL™ appeared tohave much lower titers of antibodies which were equivalent to those fromthe non-adjuvanted group.

Example 11 Effect of Adjuvants on Bactericidal Activity

Bactericidal antibody titers were assayed with sera generated against 74kD protein from O35E strain using different adjuvants. The results areshown in Table 6:

TABLE 6 The bactericidal activity of the antibodies elicited by 74 kDprotein using different adjuvants BC titers assayed against adjuvantO35E 430:345 TTA24 125-114 216-96 1230 saline <100 891 <100   <100  <100   <100   QS-21 <100 >6,400     147 374 180 500 MPL <100 <100  <100   <100   <100   113 Alum <100 <100   <100   <100   <100   <100  MPL + <100 443 151 354 216 275 Alum

Only sera generated to 74 kD protein using Stimulon™ QS-21 or the mixedadjuvant exhibited bactericidal activity towards heterologous strains.Thus, Stimulon™ QS-21 and the mixed adjuvants appeared to augmentantibody response to the conserved epitopes of the 74 kD protein.

Example 12 Immunogenicity Of The Purified 74 kD Protein

Female BALB/c mice (Taconic Farms, Germantown, N.Y.), age 6-8 weeks,were immunized subcutaneously on weeks 0 and 4 with 1 μg of purified 74kD protein formulated with 25 μg of the adjuvant Stimulon™QS-21 unlessotherwise stated. Control mice were injected with 1 μg of CPM₁₉₇ (anon-toxic variant of diphtheria toxin) and Stimulon™ QS-21. Serumsamples were collected at weeks 0 and 6. Mice were challengedintratracheally with 3.5×10⁵ CFUs of bacteria five days after the finalbleed at week 7.

The immunogenicity of the purified 74 kD protein was evaluated in thesemice in several studies which gave similar results. A representativestudy is shown in Table 7. ELISA titers were determined on pooled serumsamples from ten mice using purified protein as the detection antigen.As shown in Table 7 below, the purified 74 kD protein was immunogenic inmice. Immunization with two 1 μg doses of antigen four weeks apartelicited high antibody titers toward the purified protein by ELISA(Table 7). Antibodies elicited by the 74 kD antigen purified from eitherO35E or 430-345 strains reacted strongly against the purified 74 kDantigen from both strains, suggesting that 74 kD proteins from these twostrains were antigenically similar. When these sera were tested againstpurified 74 kD protein from TTA24 strain by ELISA, low titers weredetected (Table 7). Thus, the 74 kD protein from TTA24 strain appears todiffer antigenically from the 74 kD proteins of strains O35E or 430-345.However, when antibodies made against 74 kD protein from TTA24 strainwere assayed against the protein from O35E strain, a moderate titer wasdetected. This suggested that there is some conservation between 74 kDproteins from TTA24 and O35E strain, and the strain specific epitopesmay be more immunogenic than conserved epitopes in 74 kD protein of theO35E strain. The antisera did not react with other M. catarrhalisproteins tested, namely recombinant CopB, recombinant C/D, or purifiedUspA, as tested by ELISA or western blot (data not shown).

TABLE 7 Immunogenicity of the 74 kD Protein from Various Strains Week 6serum IgG titer elicited by 74 74 kD protein kD protein from strain fromstrain O35E 430-345 TTA24 O35E 714,085 952,314   10,580 TTA24    144   520 6,856,399 430-345 364,620 2,328,895   ND

The antisera raised against 74 kD proteins from strains O35E and 430-345in mice were tested in a whole cell ELISA against several M. catarrhalisstrains. Doses in columns 1, 3 and 5 of Table 8 below were 2×1 μgprotein; doses in columns 2 and 4 were 2×5 μg protein. The adjuvant usedwas 25 μl of Stimulon™ QS-21. These sera exhibited high whole-cell ELISAtiters against O35E and 430-345 strains, but titers to 5 other strainswere much lower (Table 8). When whole cell lysates from nine M.catarrhalis strains were resolved by 4-15% SDS-PAGE, blotted ontonitrocellulose membrane and probed with antiserum against the 74 kDprotein, only a single band at the 74 kD region was detected for all theisolates (see FIG. 5). This indicated that the titers toward wholebacteria cells were due to specific reactivity to the 74 kD protein.

TABLE 8 Immunogenicity of the 74 kD Protein from Strains O35E and430-345: IgG titers by whole cell ELISA Assay Mouse antisera to 74 kDprotein from Strain strain O35E O35E O35E 430-345 430-345 O35E 407,052 615,078 443,282 1,155,447 2,338,191 430-345 145,084  160,387 170,075  724,369   591,861 TTA24 3,458    203    532     711    2,105 ATCC252387,203    618    659    2,542    2,028 125-114 3,990    174    899   4,488    3,042 216-96 7,163    593    394    3,518   12,576 1230-3597,417  4,609  2,726    5,518    4,390

74 kD Protein from TTA24 Strain: It is clear that the 74 kD proteinsfrom strains O35E and 430-345 are antigenically similar. Antibodieselicited by 74 kD protein from these two strains reacted poorly to manyother strains of the M. catarrhalis, including TTA24 strain. Because ofthis observation, the 74 kD protein from the TTA24 strain was purifiedand evaluated in mice to determine whether it exhibited a similarpattern of conservation. As part of this experiment, a mixture of thetwo antigenically different 74 kD proteins was also examined.

BALB/c mice (10 animals/group, female, 6-8 week old at the beginning ofthe study) were immunized at weeks 0 and 4 with 5 μg of 74 kD protein(10 μg for the mixed 74 kD group) mixed with 25 μg of Stimulon™ QS-21.Whole cell ELISA titers were determined on pooled serum samples. Seraagainst 74 kD from O35E strain from a previous study was included asreference in the assay. The results are shown in Table 9:

TABLE 9 The immunogenicity of the 74 kD protein from TTA24 strain andantibody reactivity to heterologous strains IgG titer of the sera madeagainst 74 kD from strain Assay TTA24 + strain TTA24 430-345 O35E O35E26,448 568,446  958,469    430-345 51,460 731,124  376,289    1230-35928,470 68,757 59,666   TTA24 1,832,305   1,386,747   659 125-114 44,83824,971 777 216-96 97,751 86,375 4,202   ATCC25238 55,873 56,031 6,159  111-210 52,369 85,800 2,708   301-221 246,416  156,427  369 205-22110,025  6,272 598 324-171 300,613  154,140  655

The results from Table 9 indicated that antibodies elicited by 74 kDprotein from TTA24 strain exhibited very high titer against thehomologous strain and moderately high titers against 10 heterologousstrains by whole cell ELISA. Titers against heterologous strains aresignificantly higher than those of sera elicited by 74 kD proteins fromstrains O35E and 430-345. Specific reactivity of the serum to the 74 kDprotein was confirmed by western blot (FIG. 6 bottom).

As expected, a pool of 74 kD proteins from strains TTA24 and 430-345elicited a high level of antibodies against the homologous strains andthe antigenically related strain O35E. Both the ELISA and bactericidalantibody titers against eight other strains were nearly the same as thetiters elicited by 74 kD protein from TTA24 strain alone (Table 9).Specific reactivity of the antibody to the 74 kD protein was confirmedby western blot (FIG. 6 top).

In summary, the 74 kD protein from M. catarrhalis strains appears toexhibit antigenic variation. The TTA24 strain appears to express a formof the 74 kD protein that is better conserved than those of strains O35Eand 430-345. The 74 kD protein from TTA24 strain elicited antibodiesreactive to all strains of M. catarrhalis assayed. It also appearedunnecessary to use a mixture of the 74 kD proteins to generate a goodresponse.

Example 13 Enhanced Pulmonary Clearance of M. catarrhalis in Mice

To determine if immunization with purified 74 kD protein would enhancepulmonary clearance of intratracheally deposited bacteria in a murinemodel, mice immunized with the 74 kD protein prepared from strains O35Eand 430-345 were challenged with O35E or TTA24 strains of M.catarrhalis. The challenge was performed using a procedure previouslydescribed (11). In summary, 3.5×10⁵ CFUs of bacteria from amid-logarithmic culture were instilled intratracheally into the lungs ofanesthetized mice by intramuscular injection of a mixture of 2 mg ofketamine HCl (Fort Dodge Lab., Ford Dodge, Iowa) and 0.2 mg ofacepromazine maleate (Butler Co., Columbus, Ohio). Viable bacteria wererecovered from the mouse lungs six hours after challenge, and thepercentage of bacterial clearance in immunized mice was determinedrelative to the CFUs recovered from the control animals. Control animalswere immunized with CRM₁₉₇ and Stimulon™ QS-21. Statistical analysis wasperformed using the Wilcoxon rank sum test (JMP Software, SAS Institute,Cary, N.C.). A probability (p) value of less than 0.05 was consideredstatistically significant.

Eight groups of mice in several studies which were immunized withpurified 74 kD protein from the O35E strain were challenged with eitherthe O35E or the TTA24 strains. Groups 9 and 10 were immunized with 74 kDprotein from 430-345 strain and challenged with either O35E strain(group 9) or TTA24 strain (group 10). BALB/c mice (female, 6-8 weeks oldat the beginning of the study, 10 per group) were immunized at weeks 0and 4 with 1 μg of antigens mixed with 25 μg of Stimulon™ QS-21. Sera,collected 4 days before challenge, were assayed against the challengestrain by whole cell ELISA. Results are expressed as IgG endpoint titerson pooled samples. The CRM₁₉₇ control had ELISA titers of less than 100in the same assay. Mice were challenged intratracheally with 3.5×10⁵CFUs of bacteria and viable bacteria recovered from the lungs 6 hoursafter challenge. The percent clearance is the percentage of bacteriacleared from the immunized mice compared to control which were immunizedwith CRM₁₉₇ and Stimulon™ QS-21. The results of the pulmonary clearancestudy are shown in Table 10:

TABLE 10 Pulmonary clearance of M. catarrhalis in a murine challengemodel after active immunization 74 kD Challenge ELISA % Group sourcestrain titer clearance p value 1 O35E O35E 407,000 68 0.0002 2 O35E O35E168,000 70 0.0006 3 O35E O35E 102,274 63 0.0025 4 O35E O35E 1,910,012  52 0.0004 5 O35E O35E 1,193,747   57 0.0114 6 O35E O35E 235,736 470.0047 7 O35E O35E 2443,332  56 0.0003 8 O35E TTA24    140 −35   0.15  9430-345 O35E 627,148 49 0.0043 10  430-345 TTA24    337 −11   0.41  11 TTA24 O35E  26,341 29 0.318  12  TTA24 TTA24 673,754 76 0.009 

The bacterial clearance relative to control for each experiment wasconsidered statistically significant by the Wilcoxon signed rank test ifp was less than 0.05.

Relative to control mice immunized with CRM₁₉₇, enhanced pulmonaryclearance of heterologous bacteria was only seen for the mice challengedwith O35E strain (Table 10). The lack of enhanced clearance of TTA24 wasconsistent with the poor antibody reactivity toward this strain in thewhole-cell ELISA (see Table 8). Enhanced clearance of O35E, but notTTA24 strain, was also seen in mice immunized with purified 74 kDprotein from strain 430-345 (Table 10). Again this correlated with thewhole cell reactivity of the antibodies. The 74 kD protein from TTA24appears to be antigenically different from that of the O35E or 430-345strain. This may account for the inability of mice immunized with the 74kD proteins to clear this strain. Animal challenge data suggest thatimmunization with purified 74 kD protein will induce enhanced pulmonaryclearance of M. catarrhalis strains bearing antigenically similar 74 kDproteins.

Example 14 Detection of Human Serum Antibodies to the 74 kD Protein

Studies indicated that healthy adult sera contain naturally acquiredantibodies specific for M. catarrhalis (data not shown). To determine ifthey were directed toward the 74 kD protein, sera from six healthyadults were assayed for reactivity with the purified 74 kD protein fromstrains O35E and TTA24 by ELISA. All six sera had detectable titers, andtiters to 74 kD protein of the TTA24 strain were higher as shown inTable 11:

TABLE 11 Normal human sera contain naturally acquired antibodies to the74 kD protein ELISA IgG titers to 74 kD 74 kD Human serum (O35E) (TTA24)1 (H92-X)   213 2,928 2 (H89-M)   591 10,148  3 (H89-L) 1,203 5,944 4(H89-D) 1,053 5,932 5 (Kacu) 1,361 9,415 6 (Kconv) 4,683 21,592 

Specific reactivity to the 74 kD protein was seen for every serum onwestern blot (see FIG. 7). This indicated that the 74 kD protein isexpressed by the M. catarrhalis in vivo and is a target of the antibodyresponse.

Example 15 Purification of 74 kD Specific Antibodies From Human Plasma

To determine if human antibodies to the 74 kD protein recognize epitopeson the bacterial surface, 74 kD specific antibodies from the pooledplasma of two healthy adults (American Red Cross, Rochester, N.Y.) wereaffinity purified. The antibodies were precipitated by adding ammoniumsulfate to 50% saturation, resuspended and dialyzed against PBS. Anitrocellulose membrane (2×3 inches) was incubated with purified 74 kDprotein from O35E strain at 1.0 mg/ml in PBS for one hour at RT, washedtwice with PBS and residual binding sites on the membrane blocked with5% (wt/vol) dry milk in PBS for two hours at RT. The membrane was thenwashed twice with PBS, 100 mM glycine (pH 2.5) and finally with PBSbefore incubation with the dialyzed antibody preparation. Afterincubating four hours at 4° C., the membrane was washed with PBS, andthen 10 mM tris buffer (pH 8.0) containing 1 M sodium chloride to removenon-specifically bound proteins. The bound antibodies were eluted byincubating the membrane in 5 ml of 100 mM glycine (pH 2.5) for twominutes with shaking. One ml of tris-HCl (1M, pH 8.0) was immediatelyadded to the eluate to neutralize the pH. The eluted antibodies weredialyzed against PBS, aliquoted, and stored at −20° C.

As shown in FIG. 8, a western blot confirmed that the purified antibodyreacted specifically with the 74 kD protein, but did not react with theother outer membrane proteins from the whole cell lysates of O35Estrain.

ELISA end point titers are the highest antibody dilutions giving an A₄₁₅greater than three times the background when assayed against wholebacterial cells. As shown in Table 12, although the antibodies wereprepared using the 74 kD protein from O35E strain and TTA24 strain, eachreacted with five other strains by whole cell ELISA with similar titers:

TABLE 12 The whole cell reactivity of the 74 kD protein-specificantibodies purified from adult human plasma Assay IgG Titers ofAntibodies Purified Using Strain O35E TTA24 O35E 4,420 1,580 TTA24   5041,164 ATCC25238 1,325 2,230 125:114 3,015 2,130 216:96 2,859 1,1551230-359 1,960   822

The results shown in Table 12 indicated that humans mount an antibodyresponse to the conserved surface epitopes of the 74 kD protein afternatural infection.

Example 16 Inhibition of Transferrin Binding

Because transferrin may be an in vivo iron source for M. catarrhalis,the binding of antibodies to the 74 kD protein on the bacterial surfacemay interrupt the iron acquisition process. Dot blotting was used todetermine whether antibodies against the 74 kD protein could inhibittransferrin binding to the bacterial lysate. Three microliters of O35Elysate were applied to a nitrocellulose membrane. The membrane wasblocked with PBS containing 5% dry milk, followed by incubation withmouse anti-serum (1:100 diluted in PBS containing 5% dry milk) to the 74kD protein from O35E strain for two hours at room temperature. Normalmouse serum and mouse anti-serum to UspA were included as controls. Themembrane was then sequentially incubated with biotin labeledtransferrin, streptavidin-alkaline phosphatase and the enzyme substrateas described above. As depicted in FIG. 9, a reduction in transferrinbinding was observed with antibodies to the 74 kD protein. In contrast,neither a normal mouse serum nor anti-UspA serum interfered withtransferrin binding. This suggested that antibodies to the 74 kD proteinspecifically inhibit transferrin binding.

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9 1 13 PRT Moraxella catarrhalis unsure 1 uncertainties in the sequence1 Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr 1 5 10 2 17 PRTMoraxella catarrhalis unsure 1 uncertainties in the sequence 2 Xaa GlyGly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr Pro Ile Pro 1 5 10 15 Asn 320 PRT Moraxella catarrhalis unsure 1 uncertainties in the sequence 3Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr Pro Ile Pro 1 5 1015 Asn Ala Ser Gly 20 4 27 PRT Moraxella catarrhalis unsure 1uncertainties in the sequence 4 Xaa Gly Gly Ser Gly Gly Ser Asn Pro ProAla Pro Thr Pro Ile Pro 1 5 10 15 Asn Ala Ser Gly Ser Gly Asn Thr GlyAsn Thr 20 25 5 18 PRT Moraxella catarrhalis unsure 1 uncertainties inthe sequence 5 Xaa Gly Gly Ser Gly Gly Ser Asn Pro Pro Ala Pro Thr ProIle Pro 1 5 10 15 Asn Ala 6 14 PRT Moraxella catarrhalis 6 Thr Asp GluLys Asn Lys Pro Asp Gly Tyr Asn Gly Glu Tyr 1 5 10 7 15 PRT Moraxellacatarrhalis 7 Asn Gly Glu Tyr Gly His Ser Ser Glu Phe Thr Val Asn PheLys 1 5 10 15 8 14 PRT Moraxella catarrhalis 8 Thr Asp Glu Lys Asn LysPro Asp Gly Tyr Asn Gly Glu Tyr 1 5 10 9 20 PRT Moraxella catarrhalis 9Lys Ser Ile Val Ile Arg Asp Ala Asp Val Thr Gly Gly Phe Tyr Tyr 1 5 1015 Pro Asn Ala Thr 20

What is claimed is:
 1. A method of immunizing a human host against M.catarrhalis which comprises administering to said host an immunogenicamount of a vaccine composition comprising an isolated and purified 74kD protein of M. catarrhalis, wherein the 74 kD protein has: (a) amolecular weight of 74 kD as measured by mass spectrometry; (b) theamo-terinal amino acid sequence of Xaa Gly Gly Ser Gly Gly Ser Asn ProPro Ala Pro Thr (SEQ ID No:1), where the first residue is notidentified; and (c) the protein has an amino acid composition of about104 Asp+Asn residues/mole, 58 Thr residues/mole, 44 Ser residues/mole,67 Glu+Gln residues/mole, 27 Pro residues/mole, 77 Gly residues/mole, 56Ala residues/mole, 35 Val residues/mole, 6 Met residues/mole, 21 Ileresidues/mole, 40 Leu residues/mole, 25 Tyr residues/mole, 28 Pheresidues/mole, 8 His residues/mole, 72 Lys residues/mole and 21 Argresidues/mole, wherein the composition elicits a protective immuneresponse.
 2. The method of claim 1 wherein the isolated and purified 74kD protein has the amino-terminal amino acid sequence: Xaa Gly Gly SerGly Gly Ser Asn Pro Pro Ala Pro Ihr Pro Ile Pro Asn (SEQ ID NO:2). 3.The method of claim 1 wherein the isolated and purified 74 kD proteinhas the amino-terminal amino acid sequence; Xaa Gly Gly Ser Gly Gly SerAsn Pro Pro Ala Pro Thr Pro Ile Pro Asn Ala (SEQ ID NO:5).
 4. The methodof claim 1 wherein the isolated and purified 74 kD protein has theamino-terminal amino acid sequence: Xaa Gly Gly Ser Gly Gly Ser Asn ProPro Ala Pro Thr Pro Ile Pro Asn Ala Ser Gly (SEQ ID NO:3).
 5. The methodof claim 1 wherein the isolated and purified 74 kD protein has theamino-terminal amino acid sequence: Xaa Gly Gly Ser Gly Gly Ser Asn ProPro Ala Pro Thr Pro Ile Pro Asn Ala Ser Gly Ser Gly Asn Thr Gly Asn Thr(SEQ ID NO:4).
 6. The method of claim 1 which further comprisesadministering to a human host at least one additional M. catarrhalisantigen.
 7. The method of claim 6 wherein the additional M. catarrhalisantigen is selected from the group consisting of the proteins designatedCopB, UspA, OMP C/D and OMPE.
 8. The method of claim 1 wherein the 74 kDprotein is coupled to an agent which protects against another pathogenicorganism.
 9. The method of claim 1 wherein the 74 kD protein is coupledto another antigenic moiety of M. catarrhalis.
 10. The method of claim 1wherein the 74 kD protein is isolated and purified from TTA24 strain.